Novel histidine kinase

ABSTRACT

The invention provides histidine kinase polypeptides and DNA (RNA) encoding histidine kinase polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing histidine kinase polypeptides to screen for antibacterial compounds.

RELATED APPLICATIONS

[0001] This application is a Continuation-in-Part of U.S. patentapplication Ser. No. 08/878,858, filed Jun. 20, 1997.

FIELD OF THE INVENTION

[0002] This invention relates to newly identified polynucleotides andpolypeptides, and their production and uses, as well as their variants,agonists and antagonists, and their uses. In particular, in these and inother regards, the invention relates to novel polynucleotides andpolypeptides of the signal tranduction protein kinase family,hereinafter referred to as “histidine kinase”.

BACKGROUND OF THE INVENTION

[0003] The Streptococci make up a medically important genera of microbesknown to cause everal types of disease in humans, including, forexample, otitis media, conjunctivitis, pneumonia, bacteremia,meningitis, sinusitis, pleural empyema and endocarditis, and mostparticularly meningitis, such as for example infection of cerebrospinalfluid. Since its isolation more than 100 years ago, Streptococcuspneumoniae has been one of the more intensively studied microbes. Forexample, much of our early understanding that DNA is, in fact, thegenetic material was predicated on the work of Griffith and of Avery,Macleod and McCarty using this microbe. Despite the vast amount ofresearch with S. pneumoniae, many questions concerning the virulence ofthis microbe remain. It is particularly preferred to employStreptococcal genes and gene products as targets for the development ofantibiotics.

[0004] The frequency of Streptococcus pneumoniae infections has risendramatically in the past 20 years. This has been attributed to theemergence of multiply antibiotic resistant strains and an increasingpopulation of people with weakened immune systems. It is no longeruncommon to isolate Streptococcus pneumoniae strains which are resistantto some or all of the standard antibiotics. This has created a demandfor both new anti-microbial agents and diagnostic tests for thisorganism.

[0005] While certain Streptococcal factors associated with pathogenicityhave been identified, e.g., capsule polysaccharides, peptidoglycans,pneumolysins, PspA Complement factor H binding component, autolysin,neuraminidase, peptide permeases, hydrogen peroxide, IgA1 protease, thelist is certainly not complete. Moreover, very little is knownconcerning the temporal expression of such genes during infection anddisease progression in a manmmalian host. Discovering the sets of genesthe bacterium is likely to be expressing at the different stages ofinfection, particularly when an infection is established, providescritical information for the screening and characterization of novelantibacterials which can interrupt pathogenesis. In addition toproviding a fuller understanding of known proteins, such an approachwill identify previously unrecognised targets.

[0006] Many two component signal transduction systems (TCSTS) have beenidentified in bacteria (Stock, J. B., Ninfa, A. I. & Stock, A. M.(1989)Microbiol. Rev. 53, 450-490). These are involved in the bacterium'sability to monitor its surroundings and adapt to changes in itsenvironment Several of these bacterial TCSTS are involved in virulenceand bacterial pathogenesis within the host.

[0007] Histidine kinases are components of the TCSTS whichautophosphorylate a histidine residue in response to some environmentalchange. The phosphate group is then transferred to the cognate responseregulator, which affects transcription of different sets of genes. Thehistidine kinases have five short conserved amino acid sequences (Stock,J. B., Ninfa, A. J.& Stock, A. M.(1989) Microbiol. Rev. 53, 450-490,Swanson, R. V., Alex, L. A. & Simon, M. I.(1994) TIBS 19 485-491). Theseare the histidine residue, which is phosphorylated, followed afterapproximately 100 residues by a conserved asparagine residue. Afteranother 15 to 45 residues a DXGXG motif is found, followed by a FXXFmotif after another 10-20 residues. 10-20 residues further on anotherglycine motif, GXG is found. The two glycine motifs are thought to beinvolved in nucleotide binding. This family of histidine kinasesincludes PhoR protein from Bacillus subtilis. PhoR is the histidinekinase of the TCSTS which controls the genes involved in alkalinephosphatase production (Seki, T., Yoshikawa, H., Takahashi, H. & Saito,H., (1988) J. Bateriol. 170, 5935-5938.)

[0008] Response regulators are components of the TCSTS. These proteinsare phosphorylated by histidine kinases and in turn once phosphorylatedeffect the response, often through a DNA binding domain becomingactivated. The response regulators are characterized by a conservedN-terminal domain of approximately 100 amino acids. The N-terminaldomains of response regulators as well as retaining five functionallyimportant residues, corresponding to the residues D12, D13, D57, T87,K109 in CheY (Matsumura, P., Rydel, J. J., Linzmeier, R. & Vacante, D.(1984) J. Bacteriol. 160, 3641), have conserved structural features(Volz, K. (1993) Biochemistry 32, 11741-11753). The 3-dimensionalstructures of CheY from Salmonella typhimurium (Stock, A. M., Mottonen,J. M., Stock, J. B.& Schutt, ,C. E. (1989) Nature, 337, 745-749) andEscherichia coli (Volz, K. & Matsumura, P. (1991) J. Biol. Chem. 266,15511-15519) and the N-terminal domain of nitrogen regulatory protein Cfrom S. typhimurium (Volkman, B. F., Nohaile, M. J., Amy, N. K., Kustu,S. & Wemmer, D. E. (1995) Biochemistry, 34 1413-1424), are available, aswell as the secondary structure of SpoOF from Bacillus subtilis (Feher,V. A., Zapf, J. W., Hoch, J. A., Dahlquist, F. W., Whiteley, J. M. &Cavanagh, J. (1995) Protein Science, 4, 1801-1814). These structureshave an (α/β)5 fold. Several structural residues are conserved betweendifferent response regulator sequences, specifically hydrophobicresidues within the β-sheet hydrophobic core and sites from thea-helices.

[0009] Among the processes regulated by TCSTS are production ofvirulence factors, motility, antibiotic resistance and cell replication.Inhibitors of TCSTS proteins would prevent the bacterium fromestablishing and maintaining infection of the host by preventing it fromproducing the necessary factors for pathogenesis and thereby haveutility in anti-bacterial therapy

[0010] Clearly, there is a need for factors, such as the novel compoundsof the invention, that have a present benefit of being useful to screencompounds for antibiotic activity. Such factors are also useful todetermine their role in pathogenesis of infection, dysfunction anddisease. There is also a need for identification and characterization ofsuch factors and their antagonists and agonists which can play a role inpreventing, ameliorating or correcting infections, dysfunctions ordiseases.

[0011] The polypeptides of the invention have amino acid sequencehomology to a known B. subtilis phoR protein. See Seki et al.,“Nucleotide sequence of the Bacillus subtilis phoR gene”, J. Bacteriol.170 (12), 5935-5938 (1988); SWISS-PROT, accession P23545. Also seeYamada et al., “Regulation of the phosphate regulon of Escherichia coli:properties of phoR deletion mutants and subcellular localization of PhoRprotein”, Mol Gen Genet 1990 Feb;220(3):366-372; and Makino, et al.,“Nucleotide sequence of the phoR gene, a regulatory gene for thephosphate regulon of Escherichia coli”, J Mol Biol 1986 Dec5;192(3):549-556.

SUMMARY OF THE INVENTION

[0012] It is an object of the invention to provide polypeptides thathave been identified as novel histidine kinase polypeptides by homologybetween the amino acid sequence set out in Table 1 [SEQ ID NO: 2] and aknown amino acid sequence or sequences of other proteins such as B.subtilis phoR protein.

[0013] It is a further object of the invention to providepolynucleotides that encode histidine kinase polypeptides, particularlypolynucleotides that encode the polypeptide herein designated histidinekinase.

[0014] In a particularly preferred embodiment of the invention thepolynucleotide comprises a region encoding histidine kinase polypeptidescomprising the sequence set out in Table 1 [SEQ ID NO: 1] which includesa full length gene, or a variant thereof.

[0015] In another particularly preferred embodiment of the inventionthere is a novel histidine kinase protein from Streptococcus pneumoniaecomprising the amino acid sequence of Table 1 [SEQ ID NO:2], or avariant thereof.

[0016] In accordance with another aspect of the invention there isprovided an isolated nucleic acid molecule encoding a mature polypeptideexpressible by the Streptococcus pneumoniae 0100993 strain contained inthe deposited strain.

[0017] A further aspect of the invention there are provided isolatednucleic acid molecules encoding histidine kinase, particularlyStreptococcus pneumoniae histidine kinase, including mRNAs, cDNAs,genomic DNAs. Further embodiments of the invention include biologically,diagnostically, prophylactically, clinically or therapeutically usefulvariants thereof, and compositions comprising the same.

[0018] In accordance with another aspect of the invention, there isprovided the use of a polynucleotide of the invention for therapeutic orprophylactic purposes, in particular genetic immunization. Among theparticularly preferred embodiments of the invention are naturallyoccurring allelic variants of histidine kinase and polypeptides encodedthereby.

[0019] Another aspect of the invention there are provided novelpolypeptides of Streptococcus pneumoniae referred to herein as histidinekinase as well as biologically, diagnostically, prophylactically,clinically or therapeutically useful variants thereof, and compositionscomprising the same.

[0020] Among the particularly preferred embodiments of the invention arevariants of histidine kinase polypeptide encoded by naturally occurringalleles of the histidine kinase gene.

[0021] In a preferred embodiment of the invention there are providedmethods for producing the aforementioned histidine kinase polypeptides.

[0022] In accordance with yet another aspect of the invention, there areprovided inhibitors to such polypeptides, useful as antibacterialagents, including, for example, antibodies.

[0023] In accordance with certain preferred embodiments of theinvention, there are provided products, compositions and methods forassessing histidine kinase expression, treating disease, for example,otitis media, conjunctivitis, pneumonia, bacteremia, meningitis,sinusitis, pleural empyema and endocarditis, and most particularlymeningitis, such as for example infection of cerebrospinal fluid,assaying genetic variation, and administering a histidine kinasepolypeptide or polynucleotide to an organism to raise an immunologicalresponse against a bacteria, especially a Streptococcus pneumoniaebacteria.

[0024] In accordance with certain preferred embodiments of this andother aspects of the invention there are provided polynucleotides thathybridize to histidine kinase polynucleotide sequences, particularlyunder stringent conditions.

[0025] In certain preferred embodiments of the invention there areprovided antibodies against histidine kinase polypeptides.

[0026] In other embodiments of the invention there are provided methodsfor identifying compounds which bind to or otherwise interact with andinhibit or activate an activity of a polypeptide or polynucleotide ofthe invention comprising: contacting a polypeptide or polynucleotide ofthe invention with a compound to be screened under conditions to permitbinding to or other interaction between the compound and the polypeptideor polynucleotide to assess the binding to or other interaction with thecompound, such binding or interaction being associated with a secondcomponent capable of providing a detectable signal in response to thebinding or interaction of the polypeptide or polynucleotide with thecompound; and determining whether the compound binds to or otherwiseinteracts with and activates or inhibits an activity of the polypeptideor polynucleotide by detecting the presence or absence of a signalgenerated from the binding or interaction of the compound with thepolypeptide or polynucleotide.

[0027] In accordance with yet another aspect of the invention, there areprovided histidine kinase agonists and antagonists, preferablybacteriostatic or bacteriocidal agonists and antagonists.

[0028] In a further aspect of the invention there are providedcompositions comprising a histidine kinase polynucleotide or a histidinekinase polypeptide for administration to a cell or to a multicellularorganism.

[0029] Various changes and modifications within the spirit and scope ofthe disclosed invention will become readily apparent to those skilled inthe art from reading the following descriptions and from reading theother parts of the present disclosure.

GLOSSARY

[0030] The following definitions are provided to facilitateunderstanding of certain terms used frequently herein.

[0031] “Host cell” is a cell which has been transformed or transfected,or is capable of transformation or transfection by an exogenouspolynucleotide sequence.

[0032] “Identity,” as known in the art, is a relationship between two ormore polypeptide sequences or two or more polynucleotide sequences, asdetermined by comparing the sequences. hi the art, “identity” also meansthe degree of sequence relatedness between polypeptide or polynucleotidesequences, as the case may be, as determined by the match betweenstrings of such sequences. “Identity” and “similarity” can be readilycalculated by known methods, including but not limited to thosedescribed in (Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073(1988). Preferred methods to determine identity are designed to give thelargest match between the sequences tested. Methods to determineidentity and similarity are codified in publicly available computerprograms. Preferred computer program methods to determine identity andsimilarity between two sequences include, but are not limited to, theGCG program package (Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al., J. Molec.Biol. 215: 403410 (1990). The BLAST X program is publicly available fromNCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIHBethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215: 403-410(1990). As an illustration, by a polynucleotide having a nucleotidesequence having at least, for example, 95% “identity” to a referencenucleotide sequence of SEQ ID NO: 1 it is intended that the nucleotidesequence of the polynucleotide is identical to the reference sequenceexcept that the polynucleotide sequence may include up to five pointmutations per each 100 nucleotides of the reference nucleotide sequenceof SEQ ID NO: 1. In other words, to obtain a polynucleotide having anucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence may bedeleted or substituted with another nucleotide, or a number ofnucleotides up to 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence. These mutations of thereference sequence may occur at the 5′ or 3′ terminal positions of thereference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence. Analogously , by a polypeptide having an amino acidsequence having at least, for example, 95% identity to a reference aminoacid sequence of SEQ ID NO:2 is intended that the amino acid sequence ofthe polypeptide is identical to the reference sequence except that thepolypeptide sequence may include up to five amino acid alterations pereach 100 amino acids of the reference amino acid of SEQ ID NO: 2. Inother words, to obtain a polypeptide having an amino acid sequence atleast 95% identical to a reference amino acid sequence, up to 5% of theamino acid residues in the reference sequence may be deleted orsubstituted with another amino acid, or a number of amino acids up to 5%of the total amino acid residues in the reference sequence may beinserted into the reference sequence. These alterations of the referencesequence may occur at the amino or carboxy terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more contiguous groups within thereference sequence.

[0033] “Isolated” means altered “by the hand of man” from its naturalstate, i.e., if it occurs in nature, it has been changed or removed fromits original environment, or both. For example, a polynucleotide or apolypeptide naturally present in a living organism is not “isolated,”but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is “isolated”, as the term is employedherein.

[0034] “Polynucleotide(s)” generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotide(s)” include, without limitation, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions or single-, double- and triple-stranded regions,single- and double-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded, ortriple-stranded regions, or a mixture of single- and double-strandedregions. In addition, “polynucleotide” as used herein refers totriple-stranded regions comprising RNA or DNA or both RNA and DNA. Thestrands in such regions may be from the same molecule or from differentmolecules. The regions may include all of one or more of the molecules,but more typically involve only a region of some of the molecules. Oneof the molecules of a triple-helical region often is an oligonucleotide.As used herein, the term “polynucleotide(s)” also includes DNAs or RNAsas described above that contain one or more modified bases. Thus, DNAsor RNAs with backbones modified for stability or for other reasons are“polynucleotide(s)” as that term is intended herein. Moreover, DNAs orRNAs comprising unusual bases, such as inosine, or modified bases, suchas tritylated bases, to name just two examples, are polynucleotides asthe term is used herein. It will be appreciated that a great variety ofmodifications have been made to DNA and RNA that serve many usefulpurposes known to those of skill in the art. The term“polynucleotide(s)” as it is employed herein embraces such chemically,enzymatically or metabolically modified forms of polynucleotides, aswell as the chemical forms of DNA and RNA characteristic of viruses andcells, including, for example, simple and complex cells.“Polynucleotide(s)” also embraces short polynucleotides often referredto as oligonucleotide(s).

[0035] “Polypeptide(s)” refers to any peptide or protein comprising twoor more amino acids joined to each other by peptide bonds or modifiedpeptide bonds. “Polypeptide(s)” refers to both short chains, commonlyreferred to as peptides, oligopeptides and oligomers and to longerchains generally referred to as proteins. Polypeptides may contain aminoacids other than the 20 gene encoded amino acids. “Polypeptide(s)”include those modified either by natural processes, such as processingand other post-translational modifications, but also by chemicalmodification techniques. Such modifications are well described in basictexts and in more detailed monographs, as well as in a voluminousresearch literature, and they are well known to those of skill in theart. It will be appreciated that the same type of modification may bepresent in the same or varying degree at several sites in a givenpolypeptide. Also, a given polypeptide may contain many types ofmodifications. Modifications can occur anywhere in a polypeptide,including the peptide backbone, the amino acid side-chains, and theamino or carboxyl termini. Modifications include, for example,acetylation, acylation, ADP-ribosylation, amidation, covalent attachmentof flavin, covalent attachment of a heme moiety, covalent attachment ofa nucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cysteine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, glycosylation, lipid attachment, sulfation,gamma-carboxylation of glutamic acid residues, hydroxylation andADP-ribosylation, selenoylation, sulfation, transfer-RNA mediatedaddition of amino acids to proteins, such as arginylation, andubiquitination. See, for instance, PROTEINS—STRUCTURE AND MOLECULARPROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, NewYork (1993) and Wold, F., Posttranslational Protein Modifications:Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENTMODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York(1983); Seifter et al., Meth. Enymol. 182:626-646 (1990) and Rattan etal., Protein Synthesis: Posttranslational Modifications and Aging, Ann.N.Y. Acad. Sci. 663: 48-62 (1992). Polypeptides may be branched orcyclic, with or without branching. Cyclic, branched and branchedcircular polypeptides may result from post-translational naturalprocesses and may be made by entirely synthetic methods, as well.

[0036] “Variant(s)” as the term is used herein, is a polynucleotide orpolypeptide that differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. A variant of a polynucleotide or polypeptide may be a naturallyoccurring such as an allelic variant, or it may be a variant that is notknown to occur naturally. Non-naturally occurring variants ofpolynucleotides and polypeptides may be made by mutagenesis techniques,by direct synthesis, and by other recombinant methods known to skilledartisans.

DESCRIPTION OF THE INVENTION

[0037] The invention relates to novel histidine kinase polypeptides andpolynucleotides as described in greater detail below. In particular, theinvention relates to polypeptides and polynucleotides of a novelhistidine kinase of Streptococcus pneumoniae, which is related by aminoacid sequence homology to B. subtilis phoR polypeptide. The inventionrelates especially to histidine kinase having the nucleotide and aminoacid sequences set out in Table 1 [SEQ ID NO: 1] and Table 1 [SEQ ID NO:2] respectively, and to the histidine kinase nucleotide sequences of theDNA in the deposited strain and amino acid sequences encoded thereby.TABLE 1 Histidine Kinase Polynucleotide and Polypeptide Sequences (A)Sequences from Streptococcus pneumoniae histidine kinase [SEQ ID NO:1].polynucleotide sequence 5′-1 ATGAAACTAA AAAGTTATAT TTTGGTTGGA TATATTATTTCAACCCTCTT 51 AACCATTTTG GTTGTTTTTT GGGCTGTTCA AAAAATGCTG ATTGCGAAAG 101GCGAGATTTA CTTTTTGCTT GGGATGACCA TCGTTGCCAG CCTTGTCGGT 151 GCTGGGATTAGTCTCTTTCT CCTATTGCCA GTCTTTACGT CGTTGGGCAA 201 ACTCAAGGAG CATGCCAAGCGGGTAGCGGC CAAGGATTTT CCTTCAAATT 251 TGGAGGTTCA AGGTCCTGTA GAATTTCAGCAATTAGGGCA AACTTTTAAT 301 GAGATGTCCC ATGATTTGCA GGTAAGCTTT GATTCCTTGGAAGAAAGCGA 351 ACGAGAAAAG GGCTTGATGA TTGCCCAGTT GTCGCATGAT ATTAAGACCC401 CTATCACTTC GATCCAAGCG ACGGTAGAAG GGATTTTGGA TGGGATTATC 451AAGGAGTCGG AGCAAGCTCA TTATCTAGCA ACCATTGGAC GCCAGACGGA 501 GAGGCTCAATAAACTGGTTG AGGAGTTGAA TTTTTTGACC CTAAACACAG 551 CTAGAAATCA GGTGGAAACTACCAGTAAAG ACAGTATTTT TCTGGACAAG 601 CTCTTAATTG AGTGCATGAG TGAATTTCAGTTTTTGATTG AGCAGGAGAG 651 AAGAGATGTC CACTTGCAGG TAATCCCAGA GTCTGCCCGGATTGAGGGAG 701 ATTATGCTAA GCTTTCTCGT ATCTTGGTGA ATCTGGTCGA TAACGCTTTT751 AAATATTCTG CTCCAGGAAC CAAGCTGGAA GTGGTGACTA AGCTGGAGAA 801GGGCCAGCTT TCAATCAGTG TGACCGATGA AGGGCAGGGC ATTGCCCCAG 851 AGGATTTGGAAAATATTTTC AAACGCCTTT ATCGTGTCGA AACTTCGCGT 901 AACATGAAGA CAGGTGGTCATGGATTAGGA CTTGCGATTG CGCGTGAATT 951 GGCCCATCAA TTGGGTGGGG AAATCACAGTCAGCAGCCAG TACGGTCTAG 1001 GAAGTACCTT TACCCTCGTT CTCAATCTCT CTGGTAGTGAAAATAAAGCC TAA -3′ (B) histidine kinase polypeptide sequence deducedfrom the [SEQ ID NO:2]. polynucleotide sequence in this table NH₂-1MKLKSYILVG YIISTLLTIL VVFWAVQKML IAKGEIYFLL GMTIVASLVG 51 AGISLFLLLPVFTSLGKLKE HAKRVAAKDF PSNLEVQGPV EFQQLGQTFN 101 EMSHDLQVSF DSLEESEREKGLMIAQLSHD IKTPITSIQA TVEGILDGII 151 KESEQAHYLA TIGRQTERLN KLVEELNFLTLNTARNQVET TSKDSIFLDK 201 LLIECMSEFQ FLIEQERRDV HLQVIPESAR IEGDYAKLSRILVNLVDNAF 251 KYSAPGTKLE VVTKLEKGQL SISVTDEGQG IAPEDLENIF KRLYRVETSR301 NMKTGGHGLG LATARELAHQ LGGEITVSSQ YGLGSTFTLV LNLSGSENKA -COOH (C)Polynucleotide sequence embodiments [SEQ ID NO:1]. X-(R₁)_(n)-1ATGAAACTAA AAAGTTATAT TTTGGTTGGA TATATTATTT CAACCCTCTT 51 AACCATTTTGGTTGTTTTTT GGGCTGTTCA AAAAATGCTG ATTGCGAAAG 101 GCGAGATTTA CTTTTTGCTTGGGATGACCA TCGTTGCCAG CCTTGTCGGT 151 GCTGGGATTA GTCTCTTTCT CCTATTGCCAGTCTTTACGT CGTTGGGCAA 201 ACTCAAGGAG CATGCCAAGC GGGTAGCGGC CAAGGATTTTCCTTCAAATT 251 TGGAGGTTCA AGGTCCTGTA GAATTTCAGC AATTAGGGCA AACTTTTAAT301 GAGATGTCCC ATGATTTGCA GGTAAGCTTT GATTCCTTGG AAGAAAGCGA 351ACGAGAAAAG GGCTTGATGA TTGCCCAGTT GTCGCATGAT ATTAAGACCC 401 CTATCACTTCGATCCAAGCG ACGGTAGAAG GGATTTTGGA TGGGATTATC 451 AAGGAGTCGG AGCAAGCTCATTATCTAGCA ACCATTGGAC GCCAGACGGA 501 GAGGCTCAAT AAACTGGTTG AGGAGTTGAATTTTTTGACC CTAAACACAG 551 CTAGAAATCA GGTGGAAACT ACCAGTAAAG ACAGTATTTTTCTGGACAAG 601 CTCTTAATTG AGTGCATGAG TGAATTTCAG TTTTTGATTG AGCAGGAGAG651 AAGAGATGTC CACTTGCAGG TAATCCCAGA GTCTGCCCGG ATTGAGGGAG 701ATTATGCTAA GCTTTCTCGT ATCTTGGTGA ATCTGGTCGA TAACGCTTTT 751 AAATATTCTGCTCCAGGAAC CAAGCTGGAA GTGGTGACTA AGCTGGAGAA 801 GGGCCAGCTT TCAATCAGTGTGACCGATGA AGGGCAGGGC ATTGCCCCAG 851 AGGATTTGGA AAATATTTTC AAACGCCTTTATCGTGTCGA AACTTCGCGT 901 AACATGAAGA CAGGTGGTCA TGGATTAGGA CTTGCGATTGCGCGTGAATT 951 GGCCCATCAA TTGGGTGGGG AAATCACAGT CAGCAGCCAG TACGGTCTAG1001 GAAGTACCTT TACCCTCGTT CTCAATCTCT CTGGTAGTGA AAATAAAGCC -(R₂)_(n)-Y(D) Polypeptide sequence embodiments [SEQ ID NO:2]. X-(R₁)_(n)-1MKLKSYILVG YIISTLLTIL VVFWAVQKML IAKGEIYFLL GMTIVASLVG 51 AGISLFLLLPVFTSLGKLKE HAKRVAAKDF PSNLEVQGPV EFQQLGQTFN 101 EMSHDLQVSF DSLEESEREKGLMIAQLSHD IKTPITSIQA TVEGILDGII 151 KESEQAHYLA TIGRQTERLN KLVEELNFLTLNTARNQVET TSKDSIFLDK 201 LLIECMSEFQ FLIEQERRDV HLQVIPESAR IEGDYAKLSRILVNLVDNAF 251 KYSAPGTKLE VVTKLEKGQL SISVTDEGQG IAPEDLENIF KRLYRVETSR301 NMKTGGHGLG LAIARELAHQ LGGEITVSSQ YGLGSTFTLV LNLSGSENKA -(R₂)_(n)-Y(E) Sequences from Streptococcus pneumoniae histidine kinase [SEQ IDNO:3]. polynucleotide sequence (including additional 3′ and 5′untranslated sequence). 5′- 1 AGATAGAGAA ACCGAGAGGA CAAACATGAAACTAAAAAGT TATATTTTGG 51 TTGGATATAT TATTTCAACC CTCTTAACCA TTTTGGTTGTTTTTTGGGCT 101 GTTCAAAAAA TGCTGATTGC GAAAGGCGAG ATTTACTTTT TGCTTGGGAT151 GACCATCGTT GCCAGCCTTG TCGGTGCTGG GATTAGTCTC TTTCTCCTAT 201TGCCAGTCTT TACGTCGTTG GGCAAACTCA AGGAGCATGC CAAGCGGGTA 251 GCGGCCAAGGATTTTCCTTC AAATTTGGAG GTTCAAGGTC CTGTAGAATT 301 TCAGCAATTA GGGCAAACTTTTAATGAGAT GTCCCATGAT TTGCAGGTAA 351 GCTTTGATTC CTTGGAAGAA AGCGAACGAGAAAAGGGCTT GATGATTGCC 401 CAGTTGTCGC ATGATATTAA GACCCCTATC ACTTCGATCCAAGCGACGGT 451 AGAAGGGATT TTGGATGGGA TTATCAAGGA GTCGGAGCAA GCTCATTATC501 TAGCAACCAT TGGACGCCAG ACGGAGAGGC TCAATAAACT GGTTGAGGAG 551TTGAATTTTT TGACCCTAAA CACAGCTAGA AATCAGGTGG AAACTACCAG 601 TAAAGACAGTATTTTTCTGG ACAAGCTCTT AATTGAGTGC ATGAGTGAAT 651 TTCAGTTTTT GATTGAGCAGGAGAGAAGAG ATGTCCACTT GCAGGTAATC 701 CCAGAGTCTG CCCGGATTGA GGGAGATTATGCTAAGCTTT CTCGTATCTT 751 GGTGAATCTG GTCGATAACG CTTTTAAATA TTCTGCTCCAGGAACCAAGC 801 TGGAAGTGGT GACTAAGCTG GAGAAGGGCC AGCTTTCAAT CAGTGTGACC851 GATGAAGGGC AGGGCATTGC CCCAGAGGAT TTGGAAAATA TTTTCAAACG 901CCTTTATCGT GTCGAAACTT CGCGTAACAT GAAGACAGGT GGTCATGGAT 951 TAGGACTTGCGATTGCGCGT GAATTGGCCC ATCAATTGGG TGGGGAAATC 1001 ACAGTCAGCA GCCAGTACGGTCTAGGAAGT ACCTTTACCC TCGTTCTCAA 1051 TCTCTCTGGT AGTGAAAATA AAGCCTAAAACCCCTTTACA AATCCAG -3′

[0038] Deposited Materials

[0039] A deposit containing a Streptococcus pneumoniae 0100993 strainhas been deposited with the National Collections of Industrial andMarine Bacteria Ltd. (herein “NCIMB”), 23 St. Machar Drive, AberdeenAB21RY, Scotland on Apr. 11, 1996 and assigned deposit number 40794. Thedeposit was described as Streptococcus peumnoniae 0100993 on deposit. OnApr. 17, 1996, a Streptococcus peumnoniae 0100993 DNA library in E. coliwas similarly deposited with the NCIMB and assigned deposit number40800. The Streptococcus pneumoniae strain deposit is referred to hereinas “the deposited strain” or as “the DNA of the deposited strain.”

[0040] The deposited strain contains the full length histidine kinasegene. The sequence of the polynucleotides contained in the depositedstrain, as well as the amino acid sequence of the polypeptide encodedthereby, are controlling in the event of any conflict with anydescription of sequences herein.

[0041] The deposit of the deposited strain has been made under the termsof the Budapest Treaty on the International Recognition of the Depositof Micro-organisms for Purposes of Patent Procedure. The strain will beinevocably and without restriction or condition released to the publicupon the issuance of a patent. The deposited strain is provided merelyas convenience to those of skill in the art and is not an admission thata deposit is required for enablement, such as that required under 35U.S.C. §112.

[0042] A license may be required to make, use or sell the depositedstrain, and compounds derived therefrom, and no such license is herebygranted.

[0043] Polypeptides

[0044] The polypeptides of the invention include the polypeptide ofTable 1 [SEQ ID NO:2] (in particular the mature polypeptide) as well aspolypeptides and fragments, particularly those which have the biologicalactivity of histidine kinase, and also those which have at least 70%identity to the polypeptide of Table 1 [SEQ ID NO:2] or the relevantportion, preferably at least 80% identity to the polypeptide of Table 1[SEQ ID NO:2], and more preferably at least 90% similarity (morepreferably at least 90% identity) to the polypeptide of Table 1 [SEQ IDNO:2] and still more preferably at least 95% similarity (still morepreferably at least 95% identity) to the polypeptide of Table 1 [SEQ IDNO:2] and also include portions of such polypeptides with such portionof the polypeptide generally containing at least 30 amino acids and morepreferably at least 50 amino acids.

[0045] The invention also includes polypeptides of the formula set forthin Table 1 (D) wherein, at the amino terminus, X is hydrogen, and at thecarboxyl terminus, Y is hydrogen or a metal, R₁ and R₂ is any amino acidresidue, and n is an integer between 1 and 1000. Any stretch of aminoacid residues denoted by either R group, where N is greater than 1, maybe either a heteropolymer or a homopolymer, preferably a heteropolymer.

[0046] A fragment is a variant polypeptide having an amino acid sequencethat entirely is the same as part but not all of the amino acid sequenceof the aforementioned polypeptides. As with histidine kinasepolypeptides fragments may be “free-standing,” or comprised within alarger polypeptide of which they form a part or region, most preferablyas a single continuous region, a single larger polypeptide.

[0047] Preferred fragments include, for example, truncation polypeptideshaving a portion of the amino acid sequence of Table 1 [SEQ ID NO:2], orof variants thereof, such as a continuous series of residues thatincludes the amino terminus, or a continuous series of residues thatincludes the carboxyl terminus. Degradation forms of the polypeptides ofthe invention in a host cell, particularly a Streptococcus pneumoniae,are also preferred. Further preferred are fragments characterized bystructural or functional attributes such as fragments that comprisealpha-helix and alpha-helix forming regions, beta-sheet andbeta-sheet-forming regions, turn and turn-forming regions, coil andcoil-forming regions, hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions, substrate binding region, and high antigenicindex regions.

[0048] Also preferred are biologically active fragments which are thosefragments that mediate activities of histidine kinase, including thosewith a similar activity or an improved activity, or with a decreasedundesirable activity. Also included are those fragments that areantigenic or immunogenic in an animal, especially in a human.Particularly preferred are fragments comprising receptors or domains ofenzymes that confer a function essential for viability of Streptococcuspneumoniae or the ability to initiate, or maintain cause disease in anindividual, particularly a human.

[0049] Variants that are fragments of the polypeptides of the inventionmay be employed for producing the corresponding full-length polypeptideby peptide synthesis; therefore, these variants may be employed asintermediates for producing the full-length polypeptides of theinvention.

[0050] Polynucleotides

[0051] Another aspect of the invention relates to isolatedpolynucleotides, including the full length gene, that encode thehistidine kinase polypeptide having the deduced amino acid sequence ofTable 1 [SEQ ID NO:2] and polynucleotides closely related thereto andvariants thereof.

[0052] Using the information provided herein, such as the polynucleotidesequences set out in Table 1 [SEQ ID NOS:1 and 3], a polynucleotide ofthe invention encoding histidine kinase polypeptide may be obtainedusing standard cloning and screening methods, such as those for cloningand sequencing chromosomal DNA fragments from bacteria usingStreptococcus pneumoniae 0100993 cells as starting material, followed byobtaining a full length clone. For example, to obtain a polynucleotidesequence of the invention, such as the sequences given in Table 1 [SEQID NOS:1 and 3], typically a library of clones of chromosomal DNA ofStreptococcus pneumoniae 0100993 in E. coli or some other suitable hostis probed with a radiolabeled oligonucleotide, preferably a 17-mer orlonger, derived from a partial sequence. Clones carrying DNA identicalto that of the probe can then be distinguished using stringentconditions. By sequencing the individual clones thus identified withsequencing primers designed from the original sequence it is thenpossible to extend the sequence in both directions to determine the fullgene sequence. Conveniently, such sequencing is performed usingdenatured double stranded DNA prepared from a plasmid clone. Suitabletechniques are described by Maniatis, T., Fritsch, E. F. and Sambrook etal., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed.; Cold Spring HarborLaboratory Press, Cold Spring Harbor, New York (1989). See in particularScreening By Hybridization 1.90 and Sequencing Denatured Double-StrandedDNA Templates 13.70. Illustrative of the invention, the polynucleotidesset out in Table 1 [SEQ ID NOS: 1 and 3] were discovered in a DNAlibrary derived from Streptococcus pneumoniae 0100993.

[0053] The DNA sequences set out in Table 1 [SEQ ID NOS: 1 and 3]contains an open reading frame encoding a protein having about thenumber of amino acid residues set forth in Table 1 [SEQ ID NO:2] with adeduced molecular weight that can be calculated using amino acid residuemolecular weight values well known in the art. The polynucleotide of SEQID NO: 1, between nucleotide number 1 through number 1050 encodes thepolypeptide of SEQ ID NO:2. The stop codon begins at nucleotide number1051 of SEQ ID NO: 1.

[0054] Histidine Kinase of the invention is structurally related toother proteins of the signal tranduction protein kinase family, as shownby the results of sequencing the DNA encoding histidine kinase of thedeposited strain. The protein exhibits greatest homology to B. subtilisphoR protein among known proteins. Histidine kinase of Table 1 [SEQ IDNO:2] has about 32% identity over its entire length and about 51%similarity over its entire length with the amino acid sequence of B.subtilis phoR polypeptide.

[0055] The invention provides a polynucleotide sequence identical overits entire length to the coding sequence in Table 1 [SEQ ID NO:1]. Alsoprovided by the invention is the coding sequence for the maturepolypeptide or a fragment thereof, by itself as well as the codingsequence for the mature polypeptide or a fragment in reading frame withother coding sequence, such as those encoding a leader or secretorysequence, a pre-, or pro- or prepro- protein sequence. Thepolynucleotide may also contain non-coding sequences, including forexample, but not limited to non-coding 5′ and 3′ sequences, such as thetranscribed, non-translated sequences, termination signals, ribosomebinding sites, sequences that stabilize mRNA, introns, polyadenylationsignals, and additional coding sequence which encode additional aminoacids. For example, a marker sequence that facilitates purification ofthe fused polypeptide can be encoded. In certain embodiments of theinvention, the marker sequence is a hexa-histidine peptide, as providedin the pQE vector (Qiagen, Inc.) and described in Gentz et al., Proc.Natl. Acad. Sci., USA 86: 821-824 (1989), or an HA tag (Wilson et al.,Cell 37: 767 (1984). Polynucleotides of the invention also include, butare not limited to, polynucleotides comprising a structural gene and itsnaturally associated sequences that control gene expression.

[0056] A preferred embodiment of the invention is the polynucleotide ofcomprising nucleotide 1 to 1050 set forth in SEQ ID NO: 1 of Table 1which encodes the histidine kinase polypeptide.

[0057] The invention also includes polynucleotides of the formula setforth in Table 1 (C) wherein, at the 5′ end of the molecule, X ishydrogen, and at the 3′ end of the molecule, Y is hydrogen or a metal,R₁ and R₂ is any nucleic acid residue, and n is an integer between 1 and1000. Any stretch of nucleic acid residues denoted by either R group,where R is greater than 1, may be either a heteropolymer or ahomopolymer, preferably a heteropolymer.

[0058] The term “polynucleotide encoding a polypeptide” as used hereinencompasses polynucleotides that include a sequence encoding apolypeptide of the invention, particularly a bacterial polypeptide andmore particularly a polypeptide of the Streptococcus pneumoniaehistidine kinase having the amino acid sequence set out in Table 1 [SEQID NO:2]. The term also encompasses polynucleotides that include asingle continuous region or discontinuous regions encoding thepolypeptide (for example, interrupted by integrated phage or aninsertion sequence or editing) together with additional regions, thatalso may contain coding and/or non-coding sequences.

[0059] The invention further relates to variants of the polynucleotidesdescribed herein that encode for variants of the polypeptide having thededuced amino acid sequence of Table 1 [SEQ ID NO:2]. Variants that arefragments of the polynucleotides of the invention may be used tosynthesize full-length polynucleotides of the invention.

[0060] Further particularly preferred embodiments are polynucleotidesencoding histidine kinase variants, that have the amino acid sequence ofhistidine kinase polypeptide of Table 1 [SEQ ID NO:2] in which several,a few, 5 to 10, I to 5, 1 to 3, 2, 1 or no amino acid residues aresubstituted, deleted or added, in any combination. Especially preferredamong these are silent substitutions, additions and deletions, that donot alter the properties and activities of histidine kinase.

[0061] Further preferred embodiments of the invention arepolynucleotides that are at least 70% identical over their entire lengthto a polynucleotide encoding histidine kinase polypeptide having theamino acid sequence set out in Table 1 [SEQ ID NO:2], andpolynucleotides that are complementary to such polynucleotides.Alternatively, most highly preferred are polynucleotides that comprise aregion that is at least 80% identical over its entire length to apolynucleotide encoding histidine kinase polypeptide of the depositedstrain and polynucleotides complementary thereto. In this regard,polynucleotides at least 90% identical over their entire length to thesame are particularly preferred, and among these particularly preferredpolynucleotides, those with at least 95% are especially preferred.Furthermore, those with at least 97% are highly preferred among thosewith at least 95%, and among these those with at least 98% and at least99% are particularly highly preferred, with at least 99% being the morepreferred.

[0062] Preferred embodiments are polynucleotides that encodepolypeptides that retain substantially the same biological function oractivity as the mature polypeptide encoded by the DNA of Table 1 [SEQ IDNO: 1].

[0063] The invention further relates to polynucleotides that hybridizeto the herein above-described sequences. In this regard, the inventionespecially relates to polynucleotides that hybridize under stringentconditions to the herein above-described polynucleotides. As hereinused, the terms “stringent conditions” and “stringent hybridizationconditions” mean hybridization will occur only if there is at least 95%and preferably at least 97% identity between the sequences. An exampleof stringent hybridization conditions is overnight incubation at 42° C.in a solution comprising: 50% formamide, 5× SSC (150 mM NaCl, 15 mMtrisodium citrate), 50 mM sodium phosphate (pH7.6), 5× Denhardt'ssolution, 10% dextran sulfate, and 20 micrograms/ml denatured, shearedsalmon sperm DNA, followed by washing the hybridization support in 0.lxSSC at about 65° C. Hybridization and wash conditions are well known andexemplified in Sambrook et al., Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor, N.Y., (1989), particularly Chapter11 therein.

[0064] The invention also provides a polynucleotide consistingessentially of a polynucleotide sequence obtainable by screening anappropriate library containing the complete gene for a polynucleotidesequence set forth in SEQ ID NO:1 under stringent hybridizationconditions with a probe having the sequence of said polynucleotidesequence set forth in SEQ ID NO: 1 or a fragment thereof; and isolatingsaid DNA sequence. Fragments useful for obtaining such a polynucleotideinclude, for example, probes and primers described elsewhere herein.

[0065] As discussed additionally herein regarding polynucleotide assaysof the invention, for instance, polynucleotides of the invention asdiscussed above, may be used as a hybridization probe for RNA, CDNA andgenomic DNA to isolate full-length cDNAs and genomic clones encodinghistidine kinase and to isolate cDNA and genomic clones of other genesthat have a high sequence similarity to the histidine kinase gene. Suchprobes generally will comprise at least 15 bases. Preferably, suchprobes will have at least 30 bases and may have at least 50 bases.Particularly preferred probes will have at least 30 bases and will have50 bases or less.

[0066] For example, the coding region of the histidine kinase gene maybe isolated by screening using the DNA sequence provided in SEQ ID NO: 1to synthesize an oligonucleotide probe. A labeled oligonucleotide havinga sequence complementary to that of a gene of the invention is then usedto screen a library of cDNA, genomic DNA or MRNA to determine whichmembers of the library the probe hybridizes to.

[0067] The polynucleotides and polypeptides of the invention may beemployed, for example, as research reagents and materials for discoveryof treatments of and diagnostics for disease, particularly humandisease, as further discussed herein relating to polynucleotide assays.

[0068] Polynucleotides of the invention that are oligonucleotidesderived from the sequences of SEQ ID NOS: 1 and/or 2 may be used in theprocesses herein as described, but preferably for PCR, to determinewhether or not the polynucleotides identified herein in whole or in partare transcribed in bacteria in infected tissue. It is recognized thatsuch sequences will also have utility in diagnosis of the stage ofinfection and type of infection the pathogen has attained.

[0069] The invention also provides polynucleotides that may encode apolypeptide that is the mature protein plus additional amino orcarboxyl-terminal amino acids, or amino acids interior to the maturepolypeptide (when the mature form has more than one polypeptide chain,for instance). Such sequences may play a role in processing of a proteinfrom precursor to a mature form, may allow protein transport, maylengthen or shorten protein half-life or may facilitate manipulation ofa protein for assay or production, among other things. As generally isthe case in vivo, the additional amino acids may be processed away fromthe mature protein by cellular enzymes.

[0070] A precursor protein, having the mature form of the polypeptidefused to one or more prosequences may be an inactive form of thepolypeptide. When prosequences are removed such inactive precursorsgenerally are activated. Some or all of the prosequences may be removedbefore activation. Generally, such precursors are called proproteins.

[0071] In sum, a polynucleotide of the invention may encode a matureprotein, a mature protein plus a leader sequence (which may be referredto as a preprotein), a precursor of a mature protein having one or moreprosequences that are not the leader sequences of a preprotein, or apreproprotein, which is a precursor to a proprotein, having a leadersequence and one or more prosequences, which generally are removedduring processing steps that produce active and mature forms of thepolypeptide.

[0072] Vectors, Host Cells, Expression

[0073] The invention also relates to vectors that comprise apolynucleotide or polynucleotides of the invention, host cells that aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the invention.

[0074] For recombinant production, host cells can be geneticallyengineered to incorporate expression systems or portions thereof orpolynucleotides of the invention. Introduction of a polynucleotide intothe host cell can be effected by methods described in many standardlaboratory manuals, such as Davis et al., BASIC METHODS IN MOLECULARBIOLOGY, (1986) and Sambrook et al., MOLECULAR CLONING: A LABORATORYMANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989), such as, calcium phosphate transfection,DEAE-dextran mediated transfection, transvection, microinjection,cationic lipid-mediated transfection, electroporation, transduction,scrape loading, ballistic introduction and infection.

[0075] Representative examples of appropriate hosts include bacterialcells, such as streptococci, staphylococci, enterococci E. coli,streptomyces and Bacillus subtilis cells; fungal cells, such as yeastcells and Aspergillus cells; insect cells such as Drosophila S2 andSpodoptera Sf9 cells; animal cells such as CHO, COS, HeLa, C127, 3T3,BHK, 293 and Bowes melanoma cells; and plant cells.

[0076] A great variety of expression systems can be used to produce thepolypeptides of the invention. Such vectors include, among others,chromosomal, episomal and virus-derived vectors, e.g. vectors derivedfrom bacterial plasmids, from bacteriophage, from transposons, fromyeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, and vectors derived from combinations thereof,such as those derived from plasmid and bacteriophage genetic elements,such as cosmids and phagemids. The expression system constructs maycontain control regions that regulate as well as engender expression.Generally, any system or vector suitable to maintain, propagate orexpress polynucleotides and/or to express a polypeptide in a host may beused for expression in this regard. The appropriate DNA sequence may beinserted into the expression system by any of a variety of well-knownand routine techniques, such as, for example, those set forth inSambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, (supra).

[0077] For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. These signals may beendogenous to the polypeptide or they may be heterologous signals.

[0078] Polypeptides of the invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography, and lectin chromatography. Most preferably, highperformance liquid chromatography is employed for purification. Wellknown techniques for refolding protein may be employed to regenerateactive conformation when the polypeptide is denatured during isolationand or purification.

[0079] Diagnostic Assays

[0080] This invention is also related to the use of the histidine kinasepolynucleotides of the invention for use as diagnostic reagents.Detection of histidine kinase in a eukaryote, particularly a mammal, andespecially a human, will provide a diagnostic method for diagnosis of adisease. Eukaryotes (herein also “individual(s)”), particularly mammals,and especially humans, particularly those infected or suspected to beinfected with an organism comprising the histidine kinase gene may bedetected at the nucleic acid level by a variety of techniques.

[0081] Nucleic acids for diagnosis may be obtained from an infectedindividual's cells and tissues, such as bone, blood, muscle, cartilage,and skin. Genomic DNA may be used directly for detection or may beamplified enzymatically by using PCR or other amplification techniqueprior to analysis. RNA or cDNA may also be used in the same ways. Usingamplification, characterization of the species and strain of prokaryotepresent in an individual, may be made by an analysis of the genotype ofthe prokaryote gene. Deletions and insertions can be detected by achange in size of the amplified product in comparison to the genotype ofa reference sequence. Point mutations can be identified by hybridizingamplified DNA to labeled histidine kinase polynucleotide sequences.Perfectly matched sequences can be distinguished from mismatchedduplexes by RNase digestion or by differences in melting temperatures.DNA sequence differences may also be detected by alterations in theelectrophoretic mobility of the DNA fragments in gels, with or withoutdenaturing agents, or by direct DNA sequencing. See, e.g., Myers et al.,Science, 230: 1242 (1985). Sequence changes at specific locations alsomay be revealed by nuclease protection assays, such as RNase and SIprotection or a chemical cleavage method. See, e.g., Cotton et al.,Proc. Natl. Acad. Sci., USA, 85: 43974401 (1985).

[0082] Cells carrying mutations or polymorphisms in the gene of theinvention may also be detected at the DNA level by a variety oftechniques, to allow for serotyping, for example. For example, RT-PCRcan be used to detect mutations. It is particularly preferred to usedRT-PCR in conjunction with automated detection systems, such as, forexample, GeneScan. RNA or cDNA may also be used for the same purpose,PCR or RT-PCR. As an example, PCR primers complementary to a nucleicacid encoding histidine kinase can be used to identify and analyzemutations. Examples of representative primers are shown below in Table2. TABLE 2 Primers for amplification of histidine kinase polynucleotidesSEQ ID NO PRIMER SEQUENCE 4 5′-GCTTGATGATTGCCCAGTTGTC -3′ 55′-GGCCCTTCTCCAGCTTAGTCAC -3′ 7 5′-ATGAAACTAAAAAGTTATATTTTGG-3′ 85′-GGCTTTATTTTCACTACCAGAGAGA-3′

[0083] The invention further provides these primers with 1, 2, 3 or 4nucleotides removed from the 5′ and/or the 3′ end. These primers may beused for, among other things, amplifying histidine kinase DNA isolatedfrom a sample derived from an individual. The primers may be used toamplify the gene isolated from an infected individual such that the genemay then be subject to various techniques for elucidation of the DNAsequence. In this way, mutations in the DNA sequence may be detected andused to diagnose infection and to serotype and/or classify theinfectious agent.

[0084] The invention further provides a process for diagnosing, disease,preferably bacterial infections, more preferably infections byStreptococcus pneumoniae, and most preferably otitis media,conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleuralempyema and endocarditis, and most particularly meningitis, such as forexample infection of cerebrospinal fluid, comprising determining from asample derived from an individual a increased level of expression ofpolynucleotide having the sequence of Table 1 [SEQ ID NO: 1]. Increasedor decreased expression of histidine kinase polynucleotide can bemeasured using any on of the methods well known in the art for thequantation of polynucleotides, such as, for example, amplification, PCR,RT-PCR, RNase protection, Northern blotting and other hybridizationmethods.

[0085] In addition, a diagnostic assay in accordance with the inventionfor detecting over-expression of histidine kinase protein compared tonormal control tissue samples may be used to detect the presence of aninfection, for example. Assay techniques that can be used to determinelevels of a histidine kinase protein, in a sample derived from a hostare well-known to those of skill in the art. Such assay methods includeradioimmunoassays, competitive-binding assays, Western Blot analysis andELISA assays.

[0086] Antibodies

[0087] The polypeptides of the invention or variants thereof, or cellsexpressing them can be used as an immunogen to produce antibodiesimmunospecific for such polypeptides. “Antibodies” as used hereinincludes monoclonal and polyclonal antibodies, chimeric, single chain,simianized antibodies and humanized antibodies, as well as Fabfragments, including the products of an Fab immunolglobulin expressionlibrary.

[0088] Antibodies generated against the polypeptides of the inventioncan be obtained by administering the polypeptides or epitope-bearingfragments, analogues or cells to an animal, preferably a nonhuman, usingroutine protocols. For preparation of monoclonal antibodies, anytechnique known in the art that provides antibodies produced bycontinuous cell line cultures can be used. Examples include varioustechniques, such as those in Kohler, G. and Milstein, C., Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Cole etal., pg. 77-96 in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R Liss,Inc. (1985).

[0089] Techniques for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies topolypeptides of this invention. Also, transgenic mice, or otherorganisms such as other mammals, may be used to express humanizedantibodies.

[0090] Alternatively phage display technology may be utilized to selectantibody genes with binding activities towards the polypeptide eitherfrom repertoires of PCR amplified v-genes of lymphocytes from humansscreened for possessing anti-histidine kinase or from naive libraries(McCafferty, J. et al., (1990), Nature 348, 552-554; Marks, J. et al.,(1992) Biotechnology 10, 779-783). The affinity of these antibodies canalso be improved by chain shuffling (Clackson, T. et al., (1991) Nature352, 624-628).

[0091] If two antigen binding domains are present each domain may bedirected against a different epitope-termed ‘bispecific’ antibodies.

[0092] The above-described antibodies may be employed to isolate or toidentify clones expressing the polypeptides to purify the polypeptidesby affinity chromatography.

[0093] Thus, among others, antibodies against histidine kinase-polypeptide may be employed to treat infections, particularly bacterialinfections and especially otitis media, conjunctivitis, pneumonia,bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, andmost particularly meningitis, such as for example infection ofcerebrospinal fluid.

[0094] Polypeptide variants include antigenically, epitopically orimmunologically equivalent variants that form a particular aspect ofthis invention. The term “antigenically equivalent derivative” as usedherein encompasses a polypeptide or its equivalent which will bespecifically recognized by certain antibodies which, when raised to theprotein or polypeptide according to the invention, interfere with theimmediate physical interaction between pathogen and mammalian host. Theterm “immunologically equivalent derivative” as used herein encompassesa peptide or its equivalent which when used in a suitable formulation toraise antibodies in a vertebrate, the antibodies act to interfere withthe immediate physical interaction between pathogen and mammalian host.

[0095] The polypeptide, such as an antigenically or immunologicallyequivalent derivative or a fusion protein thereof is used as an antigento immunize a mouse or other animal such as a rat or chicken. The fusionprotein may provide stability to the polypeptide. The antigen may beassociated, for example by conjugation, with an immunogenic carrierprotein for example bovine serum albumin (BSA) or keyhole limpethaemocyanin (KLH). Alternatively a multiple antigenic peptide comprisingmultiple copies of the protein or polypeptide, or an antigenically orimmunologically equivalent polypeptide thereof may be sufficientlyantigenic to improve immunogenicity so as to obviate the use of acarrier.

[0096] Preferably, the antibody or variant thereof is modified to makeit less immunogenic in the individual. For example, if the individual ishuman the antibody may most preferably be “humanized”; where thecomplimentarity determining region(s) of the hybridoma-derived antibodyhas been transplanted into a human monoclonal antibody, for example asdescribed in Jones, P. et al. (1986), Nature 321, 522-525 or Tempest etal.,(1991) Biotechnology 9, 266-273.

[0097] The use of a polynucleotide of the invention in geneticimmunization will preferably employ a suitable delivery method such asdirect injection of plasmid DNA into muscles (Wolff et al., Hum MolGenet 1992, 1:363, Manthorpe et al., Hum. Gene Ther. 1963:4, 419),delivery of DNA complexed with specific protein carriers (Wu et al., JBiol Chem. 1989: 264,16985), coprecipitation of DNA with calciumphosphate (Benvenisty & Reshef, PNAS USA4, 1986:83,9551), encapsulationof DNA in various forms of liposomes (Kaneda et al., Science1989:243,375), particle bombardment (Tang et al., Nature 1992, 356:152,Eisenbraun et al., DNA Cell Biol 1993, 12:791) and in vivo infectionusing cloned retroviral vectors (Seeger et al., PNAS USA 1984:81,5849).

[0098] Antagonists and Agonists—Assays and Molecules

[0099] Polypeptides of the invention may also be used to assess thebinding of small molecule substrates and ligands in, for example, cells,cell-free preparations, chemical libraries, and natural productmixtures. These substrates and ligands may be natural substrates andligands or may be structural or functional mimetics. See, e.g., Coliganet al., Current Protocols in Immunology 1(2): Chapter 5 (1991).

[0100] The invention also provides a method of screening compounds toidentify those which enhance (agonist) or block (antagonist) the actionof histidine kinase polypeptides or polynucleotides, particularly thosecompounds that are bacteriostatic and/or bacteriocidal. The method ofscreening may involve high-throughput techniques. For example, to screenfor agonists or antagoists, a synthetic reaction mix, a cellularcompartment, such as a membrane, cell envelope or cell wall, or apreparation of any thereof, comprising histidine kinase polypeptide anda labeled substrate or ligand of such polypeptide is incubated in theabsence or the presence of a candidate molecule that may be a histidinekinase agonist or antagonist. The ability of the candidate molecule toagonize or antagonize the histidine kinase polypeptide is reflected indecreased binding of the labeled ligand or decreased production ofproduct from such substrate. Molecules that bind gratuitously, i.e.,without inducing the effects of histidine kinase polypeptide are mostlikely to be good antagonists. Molecules that bind well and increase therate of product production from substrate are agonists. Detection of therate or level of production of product from substrate may be enhanced byusing a reporter system. Reporter systems that may be useful in thisregard include but are not limited to calorimetric labeled substrateconverted into product, a reporter gene that is responsive to changes inhistidine kinase polynucleotide or polypeptide activity, and bindingassays known in the art.

[0101] Another example of an assay for histidine kinase antagonists is acompetitive assay that combines histidine kinase and a potentialantagonist with histidine kinase-binding molecules, recombinanthistidine kinase binding molecules, natural substrates or ligands, orsubstrate or ligand mimetics, under appropriate conditions for acompetitive inhibition assay. histidine kinase can be labeled, such asby radioactivity or a colorimetric compound, such that the number ofhistidine kinase molecules bound to a binding molecule or converted toproduct can be determined accurately to assess the effectiveness of thepotential antagonist.

[0102] Potential antagonists include small organic molecules, peptides,polypeptides and antibodies that bind to a polynucleotide or polypeptideof the invention and thereby inhibit or extinguish its activity.Potential antagonists also may be small organic molecules, a peptide, apolypeptide such as a closely related protein or antibody that binds thesame sites on a binding molecule, such as a binding molecule, withoutinducing histidine kinase-induced activities, thereby preventing theaction of histidine kinase by excluding histidine kinase from binding.

[0103] Potential antagonists include a small molecule that binds to andoccupies the binding site of the polypeptide thereby preventing bindingto cellular binding molecules, such that normal biological activity isprevented. Examples of small molecules include but are not limited tosmall organic molecules, peptides or peptide-like molecules. Otherpotential antagonists include antisense molecules (see Okano, J.Neurochem. 56. 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHBITORSOF GENE EXPRESSION, CRC Press, Boca Raton, Fla. (1988), for adescription of these molecules). Preferred potential antagonists includecompounds related to and variants of histidine kinase.

[0104] Each of the DNA sequences provided herein may be used in thediscovery and development of antibacterial compounds. The encodedprotein, upon expression, can be used as a target for the screening ofantibacterial drugs. Additionally, the DNA sequences encoding the aminoterminal regions of the encoded protein or Shine-Delgarno or othertranslation facilitating sequences of the respective mRNA can be used toconstruct antisense sequences to control the expression of the codingsequence of interest.

[0105] The invention also provides the use of the polypeptide,polynucleotide or inhibitor of the invention to interfere with theinitial physical interaction between a pathogen and mammalian hostresponsible for sequelae of infection. In particular the molecules ofthe invention may be used: in the prevention of adhesion of bacteria, inparticular gram positive bacteria, to mammalian extracellular matrixproteins on in-dwelling devices or to extracellular matrix proteins inwounds; to block histidine kinase protein-mediated mammalian cellinvasion by, for example, initiating phosphorylation of mammaliantyrosine kinases (Rosenshine et al., Infect. Immun. 60:2211 (1992); toblock bacterial adhesion between mammalian extracellular matrix proteinsand bacterial histidine kinase proteins that mediate tissue damage and;to block the normal progression of pathogenesis in infections initiatedother than by the implantation of in-dwelling devices or by othersurgical techniques.

[0106] This invention further provides a method of screening drugs toidentify those which interfere with i) the interaction of the histidinekinase with a response regulator, the method comprising incubating thehistidine kinase with response regulator in the presence of the drug andmeasuring the ability of the drug to block this interaction; and/or ii)the ability of the histidine kinase to autophosphorylate, the methodcomprising incubating the histidine kinase with the drug and measuringthe ability of the drug to prevent autophosphorylation.

[0107] The antagonists and agonists of the invention may be employed,for instance, to inhibit and treat otitis media, conjunctivitis,pneumonia, bacteremia, meningitis, sinusitis, pleural empyema andendocarditis, and most particularly meningitis, such as for exampleinfection of cerebrospinal fluid.

[0108]Helicobacter pylon (herein H. pylon) bacteria infect the stomachsof over one-third of the world's population causing stomach cancer,ulcers, and gastritis (International Agency for Research on Cancer(1994) Schistosomes, Liver Flukes and Helicobacter Pylori (InternationalAgency for Research on Cancer, Lyon, France;http://www.uicc.ch/ecp/ecp2904.htm). Moreover, the international Agencyfor Research on Cancer recently recognized a cause-and-effectrelationship between H. pylon and gastric adenocarcinoma, classifyingthe bacterium as a Group I (definite) carcinogen. Preferredantimicrobial compounds of the invention (agonists and antagonists ofhistidine kinase) found using screens provided by the invention,particularly broad-spectrum antibiotics, should be useful in thetreatment of H. pylon infection. Such treatment should decrease theadvent of H. pylori-induced cancers, such as gastrointestinal carcinoma.Such treatment should also cure gastric ulcers and gastritis.

[0109] Vaccines

[0110] Another aspect of the invention relates to a method for inducingan immunological response in an individual, particularly a mammal whichcomprises inoculating the individual with histidine kinase, or afragment or variant thereof, adequate to produce antibody and/or T cellimmune response to protect said individual from infection, particularlybacterial infection and most particularly Streptococcus pneumoniaeinfection. Also provided are methods whereby such immunological responseslows bacterial replication. Yet another aspect of the invention relatesto a method of inducing immunological response in an individual whichcomprises delivering to such individual a nucleic acid vector to directexpression of histidine kinase, or a fragment or a variant thereof, forexpressing histidine kinase, or a fragment or a variant thereof in vivoin order to induce an immunological response, such as, to produceantibody and/or T cell immune response, including, for example,cytokine-producing T cells or cytotoxic T cells, to protect saidindividual from disease, whether that disease is already establishedwithin the individual or not. One way of administering the gene is byaccelerating it into the desired cells as a coating on particles orotherwise. Such nucleic acid vector may comprise DNA, RNA, a modifiednucleic acid, or a DNA/RNA hybrid.

[0111] A further aspect of the invention relates to an immunologicalcomposition which, when introduced into an individual capable or havinginduced within it an immunological response, induces an immunologicalresponse in such individual to a histidine kinase or protein codedtherefrom, wherein the composition comprises a recombinant histidinekinase or protein coded therefrom comprising DNA which codes for andexpresses an antigen of said histidine kinase or protein codedtherefrom. The immunological response may be used therapeutically orprophylactically and may take the form of antibody immunity or cellularimmunity such as that arising from CTL or CD4+T cells.

[0112] A histidine kinase polypeptide or a fragment thereof may be fusedwith co-protein which may not by itself produce antibodies, but iscapable of stabilizing the first protein and producing a fused proteinwhich will have immunogenic and protective properties. Thus fusedrecombinant protein, preferably further comprises an antigenicco-protein, such as lipoprotein D from Hemophilus influenzae,Glutathione-S-transferase (GST) or beta-galactosidase, relatively largeco-proteins which solubilize the protein and facilitate production andpurification thereof. Moreover, the co-protein may act as an adjuvant inthe sense of providing a generalized stimulation of the immune system.The co-protein may be attached to either the amino or carboxy terminusof the first protein.

[0113] Provided by this invention are compositions, particularly vaccinecompositions, and methods comprising the polypeptides or polynucleotidesof the invention and immunostimulatory DNA sequences, such as thosedescribed in Sato, Y. et al. Science 273: 352 (1996).

[0114] Also, provided by this invention are methods using the describedpolynucleotide or particular fragments thereof which have been shown toencode non-variable regions of bacterial cell surface proteins in DNAconstructs used in such genetic immunization experiments in animalmodels of infection with Streptococcus pneumoniae will be particularlyuseful for identifying protein epitopes able to provoke a prophylacticor therapeutic immune response. It is believed that this approach willallow for the subsequent preparation of monoclonal antibodies ofparticular value from the requisite organ of the animal successfullyresisting or clearing infection for the development of prophylacticagents or therapeutic treatments of bacterial infection, particularlyStreptococcus pneumoniae infection, in mammals, particularly humans.

[0115] The polypeptide may be used as an antigen for vaccination of ahost to produce specific antibodies which protect against invasion ofbacteria, for example by blocking adherence of bacteria to damagedtissue. Examples of tissue damage include wounds in skin or connectivetissue caused, e.g., by mechanical, chemical or thermal damage or byimplantation of indwelling devices, or wounds in the mucous membranes,such as the mouth, mammary glands, urethra or vagina.

[0116] The invention also includes a vaccine formulation which comprisesan immunogenic recombinant protein of the invention together with asuitable carrier. Since the protein may be broken down in the stomach,it is preferably administered parenterally, including, for example,administration that is subcutaneous, intramuscular, intravenous, orintradermal. Formulations suitable for parenteral administration includeaqueous and non-aqueous sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation insotonic with the bodily fluid, preferably the blood, ofthe individual; and aqueous and non-aqueous sterile suspensions whichmay include suspending agents or thickening agents. The formulations maybe presented in unit-dose or multi-dose containers, for example, sealedampules and vials and may be stored in a freezedried condition requiringonly the addition of the sterile liquid carrier immediately prior touse. The vaccine formulation may also include adjuvant systems forenhancing the immunogenicity of the formulation, such as oil-in watersystems and other systems known in the art. The dosage will depend onthe specific activity of the vaccine and can be readily determined byroutine experimentation.

[0117] While the invention has been described with reference to certainhistidine kinase protein, it is to be understood that this coversfragments of the naturally occurring protein and similar proteins withadditions, deletions or substitutions which do not substantially affectthe immunogenic properties of the recombinant protein.

[0118] Compositions, Kits and Administration

[0119] The invention also relates to compositions comprising thepolynucleotide or the polypeptides discussed above or their agonists orantagonists. The polypeptides of the invention may be employed incombination with a non-sterile or sterile carrier or carriers for usewith cells, tissues or organisms, such as a pharmaceutical carriersuitable for administration to a subject. Such compositions comprise,for instance, a media additive or a therapeutically effective amount ofa polypeptide of the invention and a pharmaceutically acceptable carrieror excipient. Such carriers may include, but are not limited to, saline,buffered saline, dextrose, water, glycerol, ethanol and combinationsthereof. The formulation should suit the mode of administration. Theinvention further relates to diagnostic and pharmaceutical packs andkits comprising one or more containers filled with one or more of theingredients of the aforementioned compositions of the invention.

[0120] Polypeptides and other compounds of the invention may be employedalone or in conjunction with other compounds, such as therapeuticcompounds.

[0121] The pharmaceutical compositions may be administered in anyeffective, convenient manner including, for instance, administration bytopical, oral, anal, vaginal, intravenous, intraperitoneal,intramuscular, subcutaneous, intranasal or intradermal routes amongothers.

[0122] In therapy or as a prophylactic, the active agent may beadministered to an individual as an injectable composition, for exampleas a sterile aqueous dispersion, preferably isotonic.

[0123] Alternatively the composition may be formulated for topicalapplication for example in the form of ointments, creams, lotions, eyeointments, eye drops, ear drops, mouthwash, impregnated dressings andsutures and aerosols, and may contain appropriate conventionaladditives, including, for example, preservatives, solvents to assistdrug penetration, and emollients in ointments and creams. Such topicalformulations may also contain compatible conventional carriers, forexample cream or ointment bases, and ethanol or oleyl alcohol forlotions. Such carriers may constitute from about 1% to about 98% byweight of the formulation; more usually they will constitute up to about80% by weight of the formulation.

[0124] For administration to mammals, and particularly humans, it isexpected that the daily dosage level of the active agent will be from0.01 mg/kg to 10 mg/kg, typically around 1 mg/kg. The physician in anyevent will determine the actual dosage which will be most suitable foran individual and will vary with the age, weight and response of theparticular individual. The above dosages are exemplary of the averagecase. There can, of course, be individual instances where higher orlower dosage ranges are merited, and such are within the scope of thisinvention.

[0125] In-dwelling devices include surgical implants, prosthetic devicesand catheters, i.e., devices that are introduced to the body of anindividual and remain in position for an extended time. Such devicesinclude, for example, artificial joints, heart valves, pacemakers,vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinarycatheters, continuous ambulatory peritoneal dialysis (CAPD) catheters.

[0126] The composition of the invention may be administered by injectionto achieve a systemic effect against relevant bacteria shortly beforeinsertion of an in-dwelling device. Treatment may be continued aftersurgery during the in-body time of the device. In addition, thecomposition could also be used to broaden perioperative cover for anysurgical technique to prevent bacterial wound infections, especiallyStreptococcus pneumoniae wound infections.

[0127] Many orthopaedic surgeons consider that humans with prostheticjoints should be considered for antibiotic prophylaxis before dentaltreatment that could produce a bacteremia. Late deep infection is aserious complication sometimes leading to loss of the prosthetic jointand is accompanied by significant morbidity and mortality. It maytherefore be possible to extend the use of the active agent as areplacement for prophylactic antibiotics in this situation.

[0128] In addition to the therapy described above, the compositions ofthis invention may be used generally as a wound treatment agent toprevent adhesion of bacteria to matrix proteins exposed in wound tissueand for prophylactic use in dental treatment as an alternative to, or inconjunction with, antibiotic prophylaxis.

[0129] Alternatively, the composition of the invention may be used tobathe an indwelling device immediately before insertion. The activeagent will preferably be present at a concentration of 1 μg/ml to 10mg/ml for bathing of wounds or indwelling devices.

[0130] A vaccine composition is conveniently in injectable form.Conventional adjuvants may be employed to enhance the immune response. Asuitable unit dose for vaccination is 0.5-5 microgram/kg of antigen, andsuch dose is preferably administered 1-3 times and with an interval of1-3 weeks. With the indicated dose range, no adverse toxicologicaleffects will be observed with the compounds of the invention which wouldpreclude their administration to suitable individuals.

[0131] Each reference disclosed herein is incorporated by referenceherein in its entirety. Any patent application to which this applicationclaims priority is also incorporated by reference herein in itsentirety.

EXAMPLES

[0132] The examples below are carried out using standard techniques,which are well known and routine to those of skill in the art, exceptwhere otherwise described in detail. The examples are illustrative, butdo not limit the invention.

Example 1 Strain Selection, Library Production and Sequencing

[0133] The polynucleotide having the DNA sequence given in SEQ ID NO:1was obtained from a library of clones of chromosomal DNA ofStreptococcus pneumoniae in E. coli. The sequencing data from two ormore clones containing overlapping Streptococcus pneumoniae DNAs wasused to construct the contiguous DNA sequence in SEQ ID NO: 1. Librariesmay be prepared by routine methods, for example:

[0134] Methods 1 and 2 Below.

[0135] Total cellular DNA is isolated from Streptococcus pneumoniae0100993 according to standard procedures and size-fractionated by eitherof two methods.

[0136] Method 1

[0137] Total cellular DNA is mechanically sheared by passage through aneedle in order to size-fractionate according to standard procedures.DNA fragments of up to 11 kbp in size are rendered blunt by treatmentwith exonuclease and DNA polymerase, and EcoRI linkers added. Fragmentsare ligated into the vector Lambda ZapII that has been cut with EcoRI,the library packaged by standard procedures and E. coli infected withthe packaged library. The library is amplified by standard procedures.

[0138] Method 2

[0139] Total cellular DNA is partially hydrolyzed with a one or acombination of restriction enzymes appropriate to generate a series offragments for cloning into library vectors (e.g., RsaI, PalI, AluI,Bshl235I), and such fragments are size-fractionated according tostandard procedures. EcoRI linkers are ligated to the DNA and thefragments then ligated into the vector Lambda ZapII that have been cutwith EcoRI the library packaged by standard procedures, and E. coliinfected with the packaged library. The library is amplified by standardprocedures.

Example 2

[0140] The Determination of Expression During Infection of a Gene fromStreptococcus pneumoniae

[0141] Excised lungs from a 48 hour respiratory tract infection ofStreptococcus pneumoniae 0100993 in the mouse is efficiently disruptedand processed in the presence of chaotropic agents and RNAase inhibitorto provide a mixture of animal and bacterial RNA. The optimal conditionsfor disruption and processing to give stable preparations and highyields of bacterial RNA are followed by the use of hybridisation to aradiolabelled oligonucleotide specific to Streptococcus pneumoniae 16SRNA on Northern blots. The RNAase free, DNAase free, DNA and proteinfree preparations of RNA obtained are suitable for Reverse TranscriptionPCR (RT-PCR), using unique primer pairs designed from the sequence ofeach gene of Streptococcus pneumoniae 0100993.

[0142] a) Isolation of Tissue Infected with Streptococcus pneumoniae0100993 from a Mouse Animal Model of Infection (Lungs)

[0143]Streptococcus pneumoniae 0100993 is grown either on TSA/5%horseblood plates or in AGCH medium overnight, 37° C., 5% CO₂. Bacteria arethen collected and resuspended in phosphate-buffered saline to an A₆₀₀of approximately 0.4. Mice are anaesthetized with isofluorane and 50 mlof bacterial suspension (approximately 2×10⁵ bacteria) is administeredintranasally using a pipetman. Mice are allowed to recover and have foodand water ad libitum. After 48 hours, the mice are euthanized by carbondioxide overdose, and lungs are aseptically removed and snap-frozen inliquid nitrogen.

[0144] b) Isolation of Streptococcus pneumoniae 0100993 RNA fromInfected Tissue Samples

[0145] Infected tissue samples, in 2-ml cryo-strorage tubes, are removedfrom −80° C. storage into a dry ice ethanol bath. In a microbiologicalsafety cabinet, the samples are disrupted up to eight at a time whilethe remaining samples are kept frozen in the dry ice ethanol bath. Todisrupt the bacteria within the tissue sample, 50-100 mg of the tissueis transfered to a FastRNA tube containing a silica/ceramic matrix(BIO101). Immediately, 1 ml of extraction reagents (FastRNA reagents,BIO101) are added to give a sample to reagent volume ratio ofapproximately 1 to 20. The tubes are shaken in a reciprocating shaker(FastPrep FP120, BIO101) at 6000 rpm for 20-120 sec. The crude RNApreparation is extracted with chloroform/isoamyl alcohol, andprecipitated with DEPC-treated/Isopropanol Precipitation Solution(BIO101). RNA preparations are stored in this isopropanol solution at−80° C. if necessary. The RNA is pelleted (12,000g for 10 min.), washedwith 75% ethanol (v/v in DEPC-treated water), air-dried for 5-10 min,and resuspended in 0.1 ml of DEPC-treated water, followed by 5-10minutes at 55° C. Finally, after at least 1 minute on ice, 200 units ofRnasin (Promega) is added.

[0146] RNA preparations are stored at −80° C. for up to one month. Forlonger term storage, the RNA precipitate can be stored at the wash stageof the protocol in 75% ethanol for at least one year at −20° C.

[0147] Quality of the RNA isolated is assessed by running samples on 1%agarose gels. 1 x TBE gels stained with ethidium bromide are used tovisualise total RNA yields. To demonstrate the isolation of bacterialRNA from the infected tissue 1×MOPS, 2.2M formaldehyde gels are run andvacuum blotted to Hybond-N (Amersham). The blot is then hybridised witha ³²P-labelled oligonucletide probe, of sequence 5′AACTGAGACTGGCTTTAAGAGATTA 3′ [SEQ ID NO:6], specific for 16S rRNA ofStreptococcus pneumoniae. The size of the hybridising band is comparedto that of control RNA isolated from in vitro grown Streptococcuspneumoniae 0100993 in the Northern blot. Correct sized bacterial 16SrRNA bands can be detected in total RNA samples that show degradation ofthe mammalian RNA when visualised on TBE gels.

[0148] c) The removal of DNA from Streptococcus pneumoniae-derived RNA

[0149] DNA was removed from 50 microgram samples of RNA by a 30 minutetreatment at 37° C. with 20 units of RNAase-free DNAaseI (GenHunter) inthe buffer supplied in a final volume of 57 microliters.

[0150] The DNAase was inactivated and removed by treatment with TRIzolLS Reagent (Gibco BRL, Life Technologies) according to the manufacturersprotocol. DNAase treated RNA was resuspended in 100 microlitres of DEPCtreated water with the addition of Rnasin as described in Method 1.

[0151] d) The Preparation of cDNA from RNA Samples Derived from InfectedTissue

[0152] 1.5 microgram samples of DNAase treated RNA are reversetranscribed using.a SuperScript Preamplification System for First StrandcDNA Synthesis kit (Gibco BRL, Life Technologies) according to themanufacturers instructions. 75 nanogram of random hexamers is used toprime each reaction. Controls without the addition of SuperScriptIIreverse transcriptase are also run. Both +/−RT samples are treated withRNaseH before proceeding to the PCR reaction

[0153] e) The use of PCR to determine the presence of a bacterial cDNAspecies PCR reactions are set up on ice in 0.2 ml tubes by adding thefollowing components: 43 microlitres PCR Master Mix (AdvancedBiotechnologies Ltd.); I microlitre PCR primers (optimally 18-25basepairs in length and designed to possess similar annealingtemperatures), each primer at 10 mM initial concentration; and 5microlitres cDNA.

[0154] PCR reactions are run on a Perkin Elmer GeneAmp PCR System 9600as follows: 2 minutes at 94° C., then 50 cycles of 30 seconds each at94° C., 50° C. and 72° C. followed by 7 minutes at 72° C. and then ahold temperature of 20° C. (the number of cycles is optimally 30-50 todetermine the appearance or lack of a PCR product and optimally 8-30cycles if an estimation of the starting quantity of cDNA from the RTreaction is to be made); 10 microlitre aliquots are then run out on 1%1×TBE gels stained with ethidium bromide, with PCR product, if present,sizes estimated by comparison to a 100 bp DNA Ladder (Gibco BRL, LifeTechnologies). Alternatively if the PCR products are convenientlylabelled by the use of a labelled PCR primer (e.g. labelled at the 5′endwith a dye) a suitable aliquot of the PCR product is run out on apolyacrylamide sequencing gel and its presence and quantity detectedusing a suitable gel scanning system (e.g. ABI Prism™ 377 Sequencerusing GeneScan™ software as supplied by Perkin Elmer).

[0155] RT/PCR controls may include +/− reverse transcriptase reactions,16S rRNA primers or DNA specific primer pairs designed to produce PCRproducts from non-transcribed Streptococcus pneumoniae 0100993 genomicsequences.

[0156] To test the efficiency of the primer pairs they are used in DNAPCR with Streptococcus pneumoniae 0100993 total DNA. PCR reactions areset up and run as described above using approximately 1 microgram of DNAin place of the cDNA.

[0157] Primer pairs which fail to give the predicted sized product ineither DNA PCR or RT/PCR are PCR failures and as such are uninformative.Of those which give the correct size product with DNA PCR two classesare distinguished in RT/PCR: (1) Genes which are not transcribed in vivoreproducibly fail to give a product in RT/PCR; and (2) genes which aretranscribed in vivo reproducibly give the correct size product in RT/PCRand show a stronger signal in the +RT samples than the signal (if at allpresent) in -RT controls

1 8 1053 base pairs nucleic acid double linear 1 ATGAAACTAA AAAGTTATATTTTGGTTGGA TATATTATTT CAACCCTCTT AACCATTTTG 60 GTTGTTTTTT GGGCTGTTCAAAAAATGCTG ATTGCGAAAG GCGAGATTTA CTTTTTGCTT 120 GGGATGACCA TCGTTGCCAGCCTTGTCGGT GCTGGGATTA GTCTCTTTCT CCTATTGCCA 180 GTCTTTACGT CGTTGGGCAAACTCAAGGAG CATGCCAAGC GGGTAGCGGC CAAGGATTTT 240 CCTTCAAATT TGGAGGTTCAAGGTCCTGTA GAATTTCAGC AATTAGGGCA AACTTTTAAT 300 GAGATGTCCC ATGATTTGCAGGTAAGCTTT GATTCCTTGG AAGAAAGCGA ACGAGAAAAG 360 GGCTTGATGA TTGCCCAGTTGTCGCATGAT ATTAAGACCC CTATCACTTC GATCCAAGCG 420 ACGGTAGAAG GGATTTTGGATGGGATTATC AAGGAGTCGG AGCAAGCTCA TTATCTAGCA 480 ACCATTGGAC GCCAGACGGAGAGGCTCAAT AAACTGGTTG AGGAGTTGAA TTTTTTGACC 540 CTAAACACAG CTAGAAATCAGGTGGAAACT ACCAGTAAAG ACAGTATTTT TCTGGACAAG 600 CTCTTAATTG AGTGCATGAGTGAATTTCAG TTTTTGATTG AGCAGGAGAG AAGAGATGTC 660 CACTTGCAGG TAATCCCAGAGTCTGCCCGG ATTGAGGGAG ATTATGCTAA GCTTTCTCGT 720 ATCTTGGTGA ATCTGGTCGATAACGCTTTT AAATATTCTG CTCCAGGAAC CAAGCTGGAA 780 GTGGTGACTA AGCTGGAGAAGGGCCAGCTT TCAATCAGTG TGACCGATGA AGGGCAGGGC 840 ATTGCCCCAG AGGATTTGGAAAATATTTTC AAACGCCTTT ATCGTGTCGA AACTTCGCGT 900 AACATGAAGA CAGGTGGTCATGGATTAGGA CTTGCGATTG CGCGTGAATT GGCCCATCAA 960 TTGGGTGGGG AAATCACAGTCAGCAGCCAG TACGGTCTAG GAAGTACCTT TACCCTCGTT 1020 CTCAATCTCT CTGGTAGTGAAAATAAAGCC TAA 1053 350 amino acids amino acid single linear 2 Met LysLeu Lys Ser Tyr Ile Leu Val Gly Tyr Ile Ile Ser Thr Leu 1 5 10 15 LeuThr Ile Leu Val Val Phe Trp Ala Val Gln Lys Met Leu Ile Ala 20 25 30 LysGly Glu Ile Tyr Phe Leu Leu Gly Met Thr Ile Val Ala Ser Leu 35 40 45 ValGly Ala Gly Ile Ser Leu Phe Leu Leu Leu Pro Val Phe Thr Ser 50 55 60 LeuGly Lys Leu Lys Glu His Ala Lys Arg Val Ala Ala Lys Asp Phe 65 70 75 80Pro Ser Asn Leu Glu Val Gln Gly Pro Val Glu Phe Gln Gln Leu Gly 85 90 95Gln Thr Phe Asn Glu Met Ser His Asp Leu Gln Val Ser Phe Asp Ser 100 105110 Leu Glu Glu Ser Glu Arg Glu Lys Gly Leu Met Ile Ala Gln Leu Ser 115120 125 His Asp Ile Lys Thr Pro Ile Thr Ser Ile Gln Ala Thr Val Glu Gly130 135 140 Ile Leu Asp Gly Ile Ile Lys Glu Ser Glu Gln Ala His Tyr LeuAla 145 150 155 160 Thr Ile Gly Arg Gln Thr Glu Arg Leu Asn Lys Leu ValGlu Glu Leu 165 170 175 Asn Phe Leu Thr Leu Asn Thr Ala Arg Asn Gln ValGlu Thr Thr Ser 180 185 190 Lys Asp Ser Ile Phe Leu Asp Lys Leu Leu IleGlu Cys Met Ser Glu 195 200 205 Phe Gln Phe Leu Ile Glu Gln Glu Arg ArgAsp Val His Leu Gln Val 210 215 220 Ile Pro Glu Ser Ala Arg Ile Glu GlyAsp Tyr Ala Lys Leu Ser Arg 225 230 235 240 Ile Leu Val Asn Leu Val AspAsn Ala Phe Lys Tyr Ser Ala Pro Gly 245 250 255 Thr Lys Leu Glu Val ValThr Lys Leu Glu Lys Gly Gln Leu Ser Ile 260 265 270 Ser Val Thr Asp GluGly Gln Gly Ile Ala Pro Glu Asp Leu Glu Asn 275 280 285 Ile Phe Lys ArgLeu Tyr Arg Val Glu Thr Ser Arg Asn Met Lys Thr 290 295 300 Gly Gly HisGly Leu Gly Leu Ala Ile Ala Arg Glu Leu Ala His Gln 305 310 315 320 LeuGly Gly Glu Ile Thr Val Ser Ser Gln Tyr Gly Leu Gly Ser Thr 325 330 335Phe Thr Leu Val Leu Asn Leu Ser Gly Ser Glu Asn Lys Ala 340 345 350 1097base pairs nucleic acid double linear 3 AGATAGAGAA ACCGAGAGGA CAAACATGAAACTAAAAAGT TATATTTTGG TTGGATATAT 60 TATTTCAACC CTCTTAACCA TTTTGGTTGTTTTTTGGGCT GTTCAAAAAA TGCTGATTGC 120 GAAAGGCGAG ATTTACTTTT TGCTTGGGATGACCATCGTT GCCAGCCTTG TCGGTGCTGG 180 GATTAGTCTC TTTCTCCTAT TGCCAGTCTTTACGTCGTTG GGCAAACTCA AGGAGCATGC 240 CAAGCGGGTA GCGGCCAAGG ATTTTCCTTCAAATTTGGAG GTTCAAGGTC CTGTAGAATT 300 TCAGCAATTA GGGCAAACTT TTAATGAGATGTCCCATGAT TTGCAGGTAA GCTTTGATTC 360 CTTGGAAGAA AGCGAACGAG AAAAGGGCTTGATGATTGCC CAGTTGTCGC ATGATATTAA 420 GACCCCTATC ACTTCGATCC AAGCGACGGTAGAAGGGATT TTGGATGGGA TTATCAAGGA 480 GTCGGAGCAA GCTCATTATC TAGCAACCATTGGACGCCAG ACGGAGAGGC TCAATAAACT 540 GGTTGAGGAG TTGAATTTTT TGACCCTAAACACAGCTAGA AATCAGGTGG AAACTACCAG 600 TAAAGACAGT ATTTTTCTGG ACAAGCTCTTAATTGAGTGC ATGAGTGAAT TTCAGTTTTT 660 GATTGAGCAG GAGAGAAGAG ATGTCCACTTGCAGGTAATC CCAGAGTCTG CCCGGATTGA 720 GGGAGATTAT GCTAAGCTTT CTCGTATCTTGGTGAATCTG GTCGATAACG CTTTTAAATA 780 TTCTGCTCCA GGAACCAAGC TGGAAGTGGTGACTAAGCTG GAGAAGGGCC AGCTTTCAAT 840 CAGTGTGACC GATGAAGGGC AGGGCATTGCCCCAGAGGAT TTGGAAAATA TTTTCAAACG 900 CCTTTATCGT GTCGAAACTT CGCGTAACATGAAGACAGGT GGTCATGGAT TAGGACTTGC 960 GATTGCGCGT GAATTGGCCC ATCAATTGGGTGGGGAAATC ACAGTCAGCA GCCAGTACGG 1020 TCTAGGAAGT ACCTTTACCC TCGTTCTCAATCTCTCTGGT AGTGAAAATA AAGCCTAAAA 1080 CCCCTTTACA AATCCAG 1097 22 basepairs nucleic acid single linear 4 GCTTGATGAT TGCCCAGTTG TC 22 22 basepairs nucleic acid single linear 5 GGCCCTTCTC CAGCTTAGTC AC 22 25 basepairs nucleic acid single linear 6 AACTGAGACT GGCTTTAAGA GATTA 25 25base pairs nucleic acid single linear 7 ATGAAACTAA AAAGTTATAT TTTGG 2525 base pairs nucleic acid single linear 8 GGCTTTATTT TCACTACCAG AGAGA25

What is claimed is:
 1. An isolated polynucleotide comprising apolynucleotide sequence selected from the group consisting of: (a) apolynucleotide having at least a 70% identity to a polynucleotideencoding a polypeptide comprising the amino acid sequence of SEQ IDNO:2; (b) a polynucleotide having at least a 70% identity to apolynucleotide encoding the same mature polypeptide expressed by thehistidine kinase gene contained in the Streptococcus pneumoniae of thedeposited strain; (c) a polynucleotide encoding a polypeptide comprisingan amino acid sequence which is at least 70% identical to the amino acidsequence of SEQ ID NO:2; (d) a polynucleotide which is complementary tothe polynucleotide of (a), (b) or (c); and (e) a polynucleotidecomprising at least 15 sequential bases of the polynucleotide of (a),(b) or (c).
 2. The polynucleotide of claim 1 wherein the polynucleotideis DNA.
 3. The polynucleotide of claim 1 wherein the polynucleotide isRNA.
 4. The polynucleotide of claim 2 comprising the nucleic acidsequence set forth in SEQ ID NO:1.
 5. The polynucleotide of claim 2comprising nucleotide 1 to 1050 set forth in SEQ ID NO:1.
 6. Thepolynucleotide of claim 2 which encodes a polypeptide comprising theamino acid sequence of SEQ ID NO:2.
 7. A vector comprising thepolynucleotide of claim
 1. 8. A host cell comprising the vector of claim7.
 9. A process for producing a polypeptide comprising: expressing fromthe host cell of claim 8 a polypeptide encoded by said DNA.
 10. Aprocess for producing a histidine kinase polypeptide or fragmentcomprising culturing a host of claim 8 under conditions sufficient forthe production of said polypeptide or fragment.
 11. A polypeptidecomprising an amino acid sequence which is at least 70% identical to theamino acid sequence of SEQ ID NO:2.
 12. A polypeptide comprising anamino acid sequence as set forth in SEQ ID NO:2.
 13. An antibody againstthe polypeptide of claim
 11. 14. An antagonist which inhibits theactivity or expression of the polypeptide of claim
 11. 15. A method forthe treatment of an individual in need of histidine kinase polypeptidecomprising: administering to the individual a therapeutically effectiveamount of the polypeptide of claim
 11. 16. A method for the treatment ofan individual having need to inhibit histidine kinase polypeptidecomprising: administering to the individual a therapeutically effectiveamount of the antagonist of claim
 14. 17. A process for diagnosing adisease related to expression or activity of the polypeptide of claim 11in an individual comprising: (a) determining a nucleic acid sequenceencoding said polypeptide, and/or (b) analyzing for the presence oramount of said polypeptide in a sample derived from the individual. 18.A method for identifying compounds which interact with and inhibit oractivate an activity of the polypeptide of claim 11 comprising:contacting a composition comprising the polypeptide with the compound tobe screened under conditions to permit interaction between the compoundand the polypeptide to assess the interaction of a compound, suchinteraction being associated with a second component capable ofproviding a detectable signal in response to the interaction of thepolypeptide with the compound; and determining whether the compoundinteracts with and activates or inhibits an activity of the polypeptideby detecting the presence or absence of a signal generated from theinteraction of the compound with the polypeptide.
 19. A method forinducing an immunological response in a mammal which comprisesinoculating the mammal with histidine kinase polypeptide of claim 11, ora fragment or variant thereof, adequate to produce antibody and/or Tcell immune response to protect said animal from disease.
 20. A methodof inducing immunological response in a mammal which comprisesdelivering a nucleic acid vector to direct expression of histidinekinase polypeptide of claim 11, or fragment or a variant thereof, forexpressing said histidine kinase polypeptide, or a fragment or a variantthereof in vivo in order to induce an immunological response to produceantibody and/or T cell immune response to protect said animal fromdisease.